Compositions which inhibit apoptosis, methods of purifying the compositions and uses thereof

ABSTRACT

The present invention is to methods of obtaining plant-derived compositions that inhibit apoptosis, the compositions obtained thereby, compositions comprising the composition, and methods of use thereof.

This application is a divisional of application Ser. No. 08/320,155filed Oct. 7, 1994, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/158,980, filed Nov. 30, 1993, now abandoned andincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to compositions of matter which are effective ininhibiting apoptotic cell death.

BACKGROUND OF THE INVENTION

Apoptosis is a normal physiologic process that leads to individual celldeath. This process of programmed cell death is involved in a variety ofnormal and pathogenic biological events and can be induced by a numberof unrelated stimuli. Changes in the biological regulation of apoptosisalso occur during aging and are responsible for many of the conditionsand diseases related to aging. Recent studies of apoptosis have impliedthat a common metabolic pathway leading to cell death may be initiatedby a wide variety of signals, including hormones, serum growth factordeprivation, chemotherapeutic agents, ionizing radiation, and infectionby human immunodeficiency virus (HIV). Wyllie (1980) Nature 284:555-556;Kanter et al. (1984) Biochem. Biophys. Res. Commun. 118:392-399; Dukeand Cohen (1986) Lymphokine Res. 5:289-299; Tomei et al. (1988) Biochem.Biophys. Res. Commun. 155:324-331; and Kruman et al. (1991) J. Cell.Physiol. 148:267-273; Ameisen and Capron (1991) Immunol. Today12:102-105; and Sheppard and Ascher (1992) J. AIDS 5:143-147. Agentsthat affect the biological control of apoptosis thus have therapeuticutility in numerous clinical indications.

Apoptotic cell death is characterized by cellular shrinkage, chromatincondensation, cytoplasmic blebbing, increased membrane permeability andinterchromosomal DNA cleavage. Gerschenson et al. (1992) FASEB J.6:2450-2455; and Cohen and Duke (1992) Ann. Rev. Immunol. 10:267-293.

All references cited herein both supra and infra are hereby incorporatedherein by reference.

A variety of food supplements containing, in part, partially processedplant extracts have been used to ameliorate the gastrointestinaldisorders that often accompany chemotherapy, radiation and AIDS. Thesupplements generally contain carbohydrates, fat and plant proteinhydrolysates. See, e.g., Tomei and Cope et al. in Apoptosis TheMolecular Basis of Cell Death (1991) Cold Spring Harbor LaboratoryPress.

Several proteinase inhibitors derived from plant extracts haveanticarcinogenic activity. Troll et al. (1987) Adv. Cancer Res.49:265-283. The Bowman-Birk inhibitors are the best described of theseinhibitors. Birk (1985) Int. J. Pep. Pro. Res. 25:113-131. Bowman-Birkinhibitors are described as a family of disulfide bonded proteins with amolecular weight of about 8 kD which suppress cellular transformation.Chou et al. (1974) Proc. Natl. Acad. Sci. U.S.A. 71:1748-1752; Yavelowet al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:5395-5399; and Yavelow etal. (1983) Cancer Res. (Suppl.). 43:2454-2459s. Crude soybean extractscontaining Bowman-Birk inhibitors have been described. Kennedy et al.U.S. Pat. No. 4,793,996. Bowman-Birk inhibitors have also been describedimmunologically. WO 90/03574; and U.S. Pat. Nos. 4,959,310; and5,053,327. Bowman-Birk inhibitors have also been found to have activityin degranulation of macrophages. Japanese Patent No. 63-51335.

SUMMARY OF THE INVENTION

The present invention encompasses methods of obtaining compositions thatinhibit apoptosis and the compositions obtained thereby. Thecompositions are termed phytogenic apoptosis inhibitors (PAIs). Theinvention encompasses physiologically acceptable compositions suitablefor administering the PAIs in an amount sufficient to modulateapoptosis. The invention further encompasses methods of use of the PAIs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the anti-apoptotic effect of soy PAIs on confluent C3H10T1/2 cells.

FIG. 2 presents the concentration-dependent anti-apoptotic effect of soyPAIs on C3H 10T1/2 cells in exponential growth phase. In FIG. 2,represents ethanol, lot 18, ♦ represents fractionated ethanol extractlot 18, represents ethanol lot 24 and ⋄ represents fractionated ethanolextract lot 24.

FIG. 3 presents a comparison of the anti-apoptotic activity of soy PAIspurified by 50% ethanol extraction and PAIs further purified by sizefractionated size exclusion gel filtration chromatography on C3H 10T1/2cells. The data are presented as a function of trypsin inhibitoryactivity.

FIG. 4 presents the anti-apoptotic activity of various concentrations ofsoy PAIs on quiescent C3H 10T1/2 cells treated with cycloheximide toinduce apoptosis. In FIG. 4, the solid black bar represents the control,the medium gray bar represents the ethanol extract 480 μG/mL, the darkgray bar represents the ethanol extract 140 μG/mL, and the light graybar represents the ethanol extract 46 μG/mL.

FIG. 5 presents a chromatogram of monosaccharides present in PAI.

FIG. 6 depicts the effects of L/G on hypoxia-repertision of rat cardiacmyocytes.

FIG. 7 is a graph depicting the effect of soy flour extracts on foodintake after methotrexate treated rats. In FIG. 7, stands for casein, ♦stands for ProCon 2000, □ stands for Sigma Soy, ⋄ stands for AcE and ▪stands for L/G.

FIG. 8 is a graph depicting the effect of soy flour extracts onmethotrexate treated rats. In FIG. 8,  stands for casein, □ stands forAcE 10× to, ▪ stands for L/G 10× and  stands for MAcE 10×.

FIG. 9 is a graph depicting the effect of soy AcE on methotrexatetreated rats. In FIG. 9, ▪ stands for casein, □ stands for AcE 1×, stands for AcE 3× and ◯ stands for AcE 10×.

FIG. 10 is a graph depicting the effect of soy flour extracts onmethotrexate treated rats. In FIG. 10,  stands for casein, ♦ stands forL/G 1×, □ stands for L/G 3×, and ⋄ stands for L/G 10×.

FIG. 11 is a bar graph depicting the incidence of rats presenting withdiarrhea after treatment with methotrexate and various diets. In FIG.11, the open bars represent the total number of rats and thecross-hatched bars represent the number of rats with diarrhea.

FIG. 12 is a bar graph summarizing the incidence of diarrhea and weightgain in rats treated with methotrexate and various diets the first barrepresents casein, the second bar represents AcE 1×, the third barrepresents AcE 3×, the fourth bar represents AcE 10×, the fifth barrepresents L/G 1×, the sixth bar represents L/G 3×, the seventh barrepresents L/G 10×, the eighth bar represents MAcE 1×, the ninth barrepresents MAcE 3× and the tenth bar represents MAcE 10×.

FIG. 13 is a photograph depicting DNA laddering as a measure ofapoptosis in lymphocytes obtained from an HIV-infected individual.

MODES OF CARRYING OUT THE INVENTION

It has now been found that a variety of plant constituents containcomponents which, when at least partially purified, exhibit the abilityto inhibit apoptosis. This composition is readily separable fromBowman-Birk inhibitors and is distinct from other, known,therapeutically effective plant products. The composition may varyslightly in chemical constituents depending on the plant source fromwhich it is derived. The composition is referred to herein as "PAIs" asthe invention encompasses related compositions made by the methodsdescribed herein but obtained from different plant sources. Thecomposition may also be prepared synthetically by methods known in thelipid synthesis art.

PAI can be isolated from a variety of different plants and plant organs.Preferably the plants are in the leguminosae (beans and peas etc.)family, but PAIs can be isolated from other plants such as those in thesolanum (such as potatoes) and allium (such as garlic) families. PAIscan also be isolated from partially purified plant extracts including,but not limited to, molasses, lecithin, partially purified proteinconcentrates and partially purified protein hydrolysates. It is withinthe skill of one in the art, utilizing the methods described herein, todetermine whether PAIs can be isolated from a particular species ofplant, plant extract or organ within a plant.

Any plant extract or part thereof that yields the compositions issuitable for use in the present invention. The plant organs which can beutilized include, but are not limited to, stems, leaves, roots, flowers,rhizomes, and preferably, storage organs such as seeds, tubers andbulbs. Preferably, the plant part utilized is a storage organ including,but not limited to, potatoes, and garlic. Most preferably the driedseeds of legumes including, but not limited to, soybeans and peas areused for ease of processing. Although the terms "seed" and "seeds" areused herein, it should be understood that these terms encompass anyplant part which yields at least one therapeutically active PAI, or PAIthat is active in cell culture.

The invention encompasses methods for substantially purifying PAIs.Various degrees of purity can be achieved. The seeds are ground orpulverized, preferably into a powder or flour. As used herein, the termpowder refers to a ground dried plant part. The powder particles shouldbe sufficiently small enough to allow substantial surface area exposureto the various liquids to which they are exposed. Any method of grindingor pulverizing is suitable for use herein, typically grinding of seedsis accomplished by a commercial mill. PAIs are unusually heat stable,thus grinding can be done at temperatures that denature many proteins.Seed flours which are purchased commercially can also be used. Forinstance, soybean flour and various yellow and green pea flours havebeen found to contain active PAIs.

The seed powder is then delipidated by any method known in the art.Delipidation is generally accomplished by extracting the powder with asolution containing an organic solvent. Suitable organic solventsinclude, but are not limited to, acetone, carbon tetrachloride, ether,hexane and chloroform. Typically, the concentration of organic solventin the solution is from 50-100%. Preferably, the organic solvent isacetone. The concentration of organic solution used may vary withrespect to the particular solvent and the seed type; determination ofthe effective concentration is within the skill of one in the art.Preferably, in the case of acetone, the concentration is about 70%.Multiple organic extractions may also be carried out. Ratios of organicsolution to powder (volume/weight) may also vary. Although not limitedto the following range, typically the ratios are from about 2:1 to about1:20 (weight powder/volume organic solution) are employed. In the caseof acetone, a ratio of 1:5 is preferred.

Due to the stability of PAIs, the temperature and atmospheric pressureunder which delipidation takes place are largely restricted only by therespective freezing and boiling points of the organic solutionsemployed. Typically, for ease of use, the delipidation takes place atroom temperature and atmospheric pressure. The extraction time islikewise not stringent and depends largely upon the ratio of powder toorganic solution. In the case of 70% acetone extraction with a ratio of1:5, delipidation typically takes place for 30 minutes, with constantstirring.

The organic solution is then separated from the powder by any methodknown in the art. Preferably, the powder is removed by centrifugationand removal of the organic phase. Any suitable form of separation canalso be employed including, but not limited to, filtration or separationby gravity. The PAIs remain in the extracted powder.

The delipidated powder is then extracted with an aqueous solution toyield an aqueous retentate containing the PAIs. The aqueous solution canbe a buffered solution such as phosphate buffered saline (PBS) and mayalso contain up to about 80% water miscible organic solvents. Suitablewater miscible organic solvents include, but are not limited to,acetonitrile, lower alkanols, especially C₁ -C₄ alkanols such asethanol, methanol and propanol, lower alkanediols, especially C₂ -C₄alkanediols such as ethyleneglycol, and polymers of lower alkanediols,especially polyethyleneglycol. Preferably ethanol is used, mostpreferably at 50% concentration.

The ratio of aqueous solution to powder can also be varied. Theextraction ratio may be from about 1:1 to at least about 1:20 (weightpowder/volume aqueous solution). Generally, a 1:10 ratio of 50% ethanolis used. The extraction time varies also and depends on the volume ofthe aqueous solution and percentage of water miscible organic solventsused. In the case if a 1:10 volume of 50% ethanol, extraction proceedsfor 1 hour with constant mixing or stirring (or agitation).

The pH range of the aqueous solution has been found to be largelyirrelevant, ranges between 2.5 and 11 have been tested; it is thuslikely that an even broader range may be effective. Typically, for easeof use, the pH range is 7-8.

The temperature and atmospheric conditions of the aqueous extraction canvary widely and depend largely on the freezing and boiling points of theaqueous solution. Typically, the extractions are carried out at roomtemperature and atmospheric pressure. Once the aqueous extraction hastaken place, the aqueous retentate is removed for further processing.Any method of removal known in the art is suitable including but notlimited to centrifugation and filtration.

The aqueous retentate is then further purified to yield the PAI. Anywater-miscible organic solvent is removed by any method known in the artincluding but not limited to ultrafiltration, drying and dialysis.Ultrafiltration can be performed using a 10 kD molecular weight cut offto remove low molecular weight proteins such as monomers of theBowman-Birk inhibitors and organic solvents. Likewise the molecularweight cut off of the dialysis tubing can be 10 kD.

Residual organic solvent can be removed by diafiltration afterultrafiltration or by multiple changes of the dialysate, for instance,by pure water. The aqueous retentate can be stored for up to severaldays in solution and indefinitely as a lyophilized solid. Preferably,the aqueous retentate is lyophilized. Both the aqueous retentate andlyophilized retentate may be subject to further processing steps; thelyophilized retentate being resuspended in an appropriate aqueoussolution prior to further processing.

Material obtained may be further separated by passage through anymolecular weight size exclusion chromatography including, but notlimited to, Sepharose S100HR (Pharmacia Biotechnology Piscataway, N.J.U.S.A.) or BioGel P-100 (BioRad Laboratories Inc, Hercules Calif.U.S.A.) in an aqueous buffer. Suitable buffers include, but are notlimited to, 0.1 to 0.3M ammonium bicarbonate or 0.1 to 0.3M NaCl in 10to 50 mM phosphate at neutral pH.

The PAIs obtained from size exclusion chromatography are found in thevoid volume and have an apparent molecular weight of greater than 80 kD.The material found in this fraction may be resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducingconditions and contains several proteins. Staining with Coomassie Blueindicates the presence of 6-8 proteins of a molecular weight range from18 to 68 kD. Analysis by thin layer chromatography demonstrates thepresence of several lipid-type compounds.

The fractions eluted from the chromatography with the greatestabsorbance at 280 nm are coincident with the fractions containing thegreatest biological activity. The biological activity separates from lowmolecular weight material and elutes in the void volume at a positionthat is consistent with that of a molecular weight of greater than 80 kDor an aggregate. This material may be concentrated, dialyzed,lyophilized and stored indefinitely in a lyophilized form.

Solubilized PAIs can be precipitated with acetone at a concentration of70% or more. However, treatment with various agents, including strongacids, destroy their activity. For instance, trifluoroacetic acid,hydrochloric acid, trichloroacetic acid and phenol destroy theiractivity. However, pH levels as low as 2.5 do not destroy activity, as1% acetic acid does not affect the activity of PAIs.

The PAIs can be further isolated by extracting a freeze dried highmolecular weight fraction obtained from defatted and ethanol extractedseed flour into a single phase mixture of chloroform:methanol:water(4:8:3). This is most conveniently done by dissolving the dried materialin the water fraction, then adding methanol followed by chloroform,mixing and removing the precipitate. This extraction yields aglycolipid/lipid/phospholipid fraction which retains PAI activity.

For example, 0.1 gEQ (the amount derived from 0.1 g of startingmaterial) of the high molecular weight fraction (FACE) was dissolved in7.5 ml of water and with constant mixing, 20 ml of methanol was addedfollowed by 10 ml of chloroform. The insoluble material was removed bycentrifugation (8,000×g×10 min) and the PAI reclaimed from the solutionby rotary evaporation to remove the organic solvents and freeze-dryingto remove the water. The fraction obtained has been termed the L/Gfraction. The carbohydrate composition of the L/G fraction consists ofarabinose and galactose in a 3:2 ratio with fucose, rhamnose,glucosamine, glucose and mannose all present as minor constituents.

The L/G fraction can be further separated on the basis of its solubilityin a mixture of chloroform:methanol and resolved by silicachromatography either in a chromatography column or by two-dimensionalthin-layer chromatography (TLC) plate format.

Typically, soy flour, or fractions thereof, is extracted by suspendingin water (20% weight per volume in the case of flour) and adding twovolumes of methanol and one volume of chloroform. This is a single phaseand is stirred/mixed at room temperature for thirty minutes to one hour.To this mixture is added one volume of chloroform, mixed, and one volumeof water. This forms two phases and the phases are separated bycentrifugation or a separating funnel after first removing any solids(if flour has been used as the starting material). The activity is inthe organic (bottom) phase.

The material is then subjected to a preliminary chromatography step onsilicic acid, i.e., a Mallinckrodt SiO₂.xH₂ O 100 mesh powder. Theactive material is loaded in chloroform and washed with chloroform or achloroform: methanol mixture of 90:10 or 80:20 and eluted with methanolor CHCl₃ :MeOH (10:90 or 20:80).

The active material can be further separated by HPLC chromatography on asilica column such as Dynamax 60A Si column from Rainin Instrument Co.,Inc. The gradient used to elute the active material is from 95:3:2 0.05acetonitrile:methanol:water: ammonia hydroxide to 65:21:14 0.35acetonitrile:methanol:water:ammonia hydroxide. The elution profile ismonitored at 205 nm.

The active fractions obtained may be further separated on the basis oftheir hydrophobicity, for instance by HPLC on a C18 column (Dynamax 60A,Rainin Instrument Co. Inc.) in methanol. The material is elutedisocratically in 90:10 methanol:5 mM NaPO₄ pH 7.4.

A variety of methods are known in the art for purifying and analyzinglipids. Any method known in the art may be used in the practice of thepresent invention provided it results in purification of an activefraction. For review, see Bligh and Dyer (1959) Can. J. Biochem.Physiol. 37:911-917; Patton et al. (1982) J. Lipid Res. 23:190-196;Jungalwala (1985) Recent Developments in Techniques for PhospholipidAnalysis, in Phospholipids in Nervous Tissues (ed. Eichberg) John Wileyand Sons, pp. 1-44; Hamilton et al. (1992) in the series, A PracticalApproach (Richwood et al. eds.) IRL Press at Oxford University Press;and Kates (1986) Techniques of Lipidology: Isolation, Analysis andIdentification in Laboratory Techniques in Biochemistry and MolecularBiology (Burdon et al. eds.) Elsevier.

The in vitro apoptosis inhibitory activity of the PAIs appears to belargely limited to actively proliferating cells; quiescent cells appearto be relatively unaffected.

The active component of the PAIs is highly stable in the presence ofproteases. For instance, the PAIs have been treated with trypsin,chymotrypsin, papain, elastase, subtilisin, and proteinase K underconditions suitable for proteolysis but the proteases have no effect ontheir activity. The high molecular weight form of the PAIs are thusproteinase-resistant.

Carbohydrate analysis can be performed, using the reagents of the OxfordGlycoSystems N-Glycan Recovery Kit as described by the manufacturersinstructions, or similar protocol. The N-Glycan Recovery Kit is based onthe use of anhydrous hydrazine to cleave N-Glycosidic bonds as reportedby Takasaki et al. (1982) Meth. Enzymol. 83:263-268. In order to analyzethe active constituent(s) of PAIs, the partially purified PAIs aretreated in accordance with the manufacturer's instructions in thefollowing manner: 1) lyophilized to remove all traces of water; 2)treated with anhydrous hydrazine at 95° C. for 5 hours to cleaveN-glycosidic bonds; 3) subjected to chromatography to remove unreactedhydrazine and peptide material; 4) treated with acetic anhydride tore-N-acetylate any de-N-acetylated amino groups; 5) a trace of Cu²⁺ ionis added to the sample to convert the acetohydrazone derivatives of thesample to unreduced oligosaccharides; 6) subjected to mixed-bedion-exchange chromatography to remove Cu²⁺ ion and monoacetyl hydrazine;and 7) active constituent(s) of PAI are recovered in a dilute solutionof acetic acid from a sample which presumably contains unreducedN-glycans, substantially free of peptide material.

One or more active components of PAIs can be co-purified with theN-Glycans of a PAI sample, when processed and isolated by an OxfordGlycoSystems N-Glycan Recovery Kit or similar method. When partiallypurified samples of PAI are processed and purified using OxfordGlycoSystems Kits for N-glycan recovery, O-glycan recovery, anddeglycosylation of proteins, only samples processed and purified usingthe N-Glycan Recovery Kit retain anti-apoptotic activity as shown inExample 6. Further, either the active component(s) of PAI all reside inthe N-Glycan purified fraction or the activity of other activecomponents is destroyed by the processing and purification conditions ofthe Oxford GlycoSystems Glycofree™ Deglycosylation Kit and O-GlycanRecovery Kit.

The invention further comprises therapeutic compositions comprisingsubstantially purified PAIs. The level of purity necessary for thecomposition can be determined empirically and is within the skill of onein the art. The compositions are suitable for use in a variety ofdisorders, as described below, and in both human and veterinaryapplications.

The activity of the PAIs, as well as active fractions thereof obtainedduring the purification method can be measured in any anti-apoptosisassay known in the art. These include, but are not limited to, the serumdeprivation of the C3H 10T1/2 cell assay described in detail in commonlyowned PCT/US94/04942 claiming priority to Apr. 30, 1993 which is thepreferred assay method, as well as the methods described in Examples 3and 4. Furthermore, in vivo apoptosis inhibition may be measured by anymethod known in the art.

In general, PAIs are pharmaceutically acceptable due to their lowtoxicity in the therapeutic dosage range, stability and ability to beincorporated into a wide variety of vehicles for numerous routes ofadministration. The PAIs can be administered alone or in combinationwith other pharmaceutically effective agents including, but not limitedto, antibiotics, wound healing agents, antioxidants and othertherapeutic agents. Suitable antibiotics include, but are not limitedto, ampicillin, tetracycline, chloramphenicol and penicillin. Suitablewound healing agents include, but are not limited to, transforminggrowth factors (TGF-βs), epidermal growth factors (EGFs), fibroblastgrowth factors (FGFs) and platelet-derived growth factors (PDGFs).Suitable antioxidants include, but are not limited to, vitamins C and E.

The compositions contain at least a therapeutically effective amount ofat least one PAI and a physiologically acceptable carrier. Aphysiologically acceptable carrier is one that does not cause an adversephysical reaction upon administration and one in which PAIs aresufficiently soluble to deliver a therapeutically effective amount ofthe compound. The therapeutically effective amount depends in part uponthe manner of introduction and the indication to be treated and othercriteria evident to ordinary skill of one in the art. Typically, atherapeutically effective amount is one sufficient to modulate apoptosisin the condition being treated as evidenced by amelioration of thesymptoms. Typically, a therapeutically effective amount is from about0.5-100% by weight of at least one PAI. The route(s) of administrationuseful in a particular indication are discussed below and are well knownto one of skill in the art.

Routes of administration include, but are not limited to, topical,transdermal, parenteral, gastrointestinal, transbronchial andtransalveolar. Topical administration is accomplished via a topicallyapplied cream, gel, rinse, etc. containing therapeutically effectiveamounts of PAIs. Transdermal administration is accomplished byapplication of a cream rinse, gel, etc. capable of allowing the PAIs topenetrate the skin and enter the blood stream. Parenteral routes ofadministration include, but are not limited to, direct injection such asintravenous, intramuscular, intraperitoneal or subcutaneous injection.Gastrointestinal routes of administration include, but are not limitedto, ingestion and rectal. Transbronchial and transalveolar routes ofadministration include, but are not limited to, inhalation, either viathe mouth or intranasally and direct injection into an airway, such asthrough a tracheotomy.

While the PAIs can be topically administered alone, it may be desirableto administer them in admixture with a topical pharmaceutically orcosmetically acceptable carrier.

"Topical pharmaceutically acceptable carrier" as used herein is anysubstantially non-toxic carrier conventionally usable for topicaladministration of pharmaceuticals in which the PAIs will remain stableand bioavailable when applied directly to skin or mucosal surfaces. Forexample, the PAIs can be dissolved in a liquid, dispersed or emulsifiedin a medium in a conventional manner to form a liquid preparation ormixed with a semi-solid (gel) or solid carrier to form a paste, powder,ointment, cream, lotion or the like.

Suitable topical pharmaceutically acceptable carriers include water,petroleum jelly (vaseline), petrolatum, mineral oil, vegetable oil,animal oil, organic and inorganic waxes, such as microcrystalline,paraffin and ozocerite wax, natural polymers, such as xanthanes,gelatin, cellulose, collagen, starch, or gum arabic, synthetic polymers,such as discussed below, alcohols, polyols, and the like. The carriermay be a water miscible carrier composition that is substantiallymiscible in water. Such water miscible topical pharmaceuticallyacceptable carrier composition can include those made with one or moreappropriate ingredients set forth above but can also include sustainedor delayed release carriers, including water containing, waterdispersible or water soluble compositions, such as liposomes,microsponges, microspheres or microcapsules, aqueous base ointments,water-in-oil or oil-in-water emulsions, gels or the like.

In one embodiment of the invention, the topical pharmaceuticallyacceptable carrier comprises a sustained release or delayed releasecarrier. The carrier is any material capable of sustained or delayedrelease of the PAIs to provide a more efficient administration resultingin one or more of less frequent and/or decreased dosage of the PAIs,ease of handling, and extended or delayed effects on dermatologicconditions. The carrier is capable of releasing the PAIs when exposed toany oily, fatty, waxy, or moist environment on the area being treated orby diffusing or by release dependent on the degree of loading of thePAIs to the carrier in order to obtain release of the PAIs. Non-limitingexamples of such carriers include liposomes, microsponges, microspheres,or microcapsules of natural and synthetic polymers and the like.Examples of suitable carriers for sustained or delayed release in amoist environment include gelatin, gum arabic, xanthane polymers; bydegree of loading include lignin polymers and the like; by oily, fattyor waxy environment include thermoplastic or flexible thermoset resin orelastomer including thermoplastic resins such as polyvinyl halides,polyvinyl esters, polyvinylidene halides and halogenated polyolefins,elastomers such as brasiliensis, polydienes, and halogenated natural andsynthetic rubbers, and flexible thermoset resins such as polyurethanes,epoxy resins and the like. Preferably, the sustained or delayed releasecarrier is a liposome, microsponge, microsphere or gel.

The compositions used in the method of treating dermatologic conditionsof the invention are applied directly to the areas to be treated. Whilenot required, it is desirable that the topical composition maintain thePAIs at the desired location for about 24 to 48 hours.

If desired, one or more additional ingredients conventionally found intopical pharmaceutical or cosmetic compositions can be included with thecarrier: such as a moisturizer, humectants, odor modifier, buffer,pigment, preservative, vitamins such as A, C and E, emulsifier,dispersing agent, wetting agent, odor-modifying agent, gelling agents,stabilizer, propellant, antimicrobial agents, sunscreen, enzymes and thelike. Those of skill in the art of topical pharmaceutical formulationscan readily select the appropriate specific additional ingredients andamounts thereof. Suitable non-limiting examples of additionalingredients include superoxide dismutase, stearyl alcohol, isopropylmyristate, sorbitan monooleate, polyoxyethylene stearate, propyleneglycol, water, alkali or alkaline earth lauryl sulfate, methylparaben,octyl dimethyl-p-amino benzoic acid (Padimate O), uric acid, reticulin,polymucosaccharides, hyaluronic acids, aloe vera, lecithin,polyoxyethylene sorbitan monooleate, vitamin A or C, tocopherol (vitaminE), alpha-hydroxy of alpha-keto acids such as pyruvic, lactic orglycolic acids, or any of the topical ingredients disclosed in U.S. Pat.Nos. 4,340,586, 4,695,590, 4,959,353 or 5,130,298 and 5,140,043.

Because dermatologic conditions to be treated may be visible, thetopical carrier can also be a topical cosmetically acceptable carrier.By "topical cosmetically acceptable carrier" as used herein is meant anysubstantially non-toxic carrier conventionally usable for topicaladministration of cosmetics in which the PAIs will remain stable andbioavailable when applied directly to the skin surface. Suitablecosmetically acceptable carriers are known to those of skill in the artand include, but are not limited to, cosmetically acceptable liquids,creams, oils, lotions, ointments, gels, or solids, such as conventionalcosmetic night creams, foundation creams, suntan lotions, sunscreens,hand lotions, make-up and make-up bases, masks and the like. Thus, to asubstantial extent topical cosmetically acceptable carriers andpharmaceutically acceptable carriers are similar, if not oftenidentical, in nature so that most of the earlier discussion onpharmaceutically acceptable carriers also applies to cosmeticallyacceptable carriers. The compositions can contain other ingredientsconventional in cosmetics including perfumes, estrogen, vitamins A, C orE, alpha-hydroxy or alpha-keto acids such as pyruvic, lactic or glycolicacids, lanolin, vaseline, aloe vera, methyl or propyl paraben, pigmentsand the like.

The effective amount of the PAIs in the compositions used to treatdermatologic conditions or diseases can vary depending on such factorsas condition of the skin, age of the skin, the particular PAI or degreeof the purity of the PAIs employed, the type of formulation and carrieringredients used, frequency of administration, overall health of theindividual being treated and the like. The precise amount for anyparticular patient use can be determined by those of skill in thepharmaceutical art taking into consideration these factors and thepresent disclosure. Preferably the composition is administered in atleast two doses and no more than about six doses per day, or less when asustained or delayed release form is used.

The compositions for topical administration usually contain from about0.0001% to about 90% by weight of the PAIs compared to the total weightof the composition, preferably from about 0.5% to about 20% by weight ofthe PAIs to composition, and especially from about 2% to about 5% byweight of the PAIS to the composition.

The topical composition is administered by applying a coating or layerto the skin or mucosal area desired to be treated. As a practical matterof convenience, the applied material is rubbed into the area.Applications need not be rubbed into the skin and the layer or coatingcan be left on the skin overnight.

The present invention provides compositions suitable for transdermaladministration including, but not limited to, pharmaceuticallyacceptable lotions, suspensions, oils, creams, ointments, rinses, gelsand liposomal carriers suspended in a suitable vehicle in which atherapeutically effective amount of PAIs has been admixed. Suchcompositions are applied directly to the skin or incorporated into aprotective carrier such as a transdermal device (so-called "patch").Examples of suitable creams, ointments etc. can be found, for instance,in the Physician's Desk Reference. Examples of suitable transdermaldevices are described, for instance, in U.S. Pat. No. 4,818,540 (Chienet al.).

The present invention includes compositions of PAIs suitable forparenteral administration including, but not limited to,pharmaceutically acceptable sterile isotonic solutions. Such solutionsinclude, but are not limited to, saline and phosphate buffered salinefor intravenous, intramuscular, intraperitoneal or subcutaneousinjection of PAIs.

The present invention includes compositions of PAIs suitable forgastrointestinal administration including, but not limited to,pharmaceutically acceptable powders, pills or liquids for ingestion andsuppositories for rectal administration.

The present invention includes compositions of PAIs suitable fortransbronchial and transalveolar administration including, but notlimited to, various types of pharmaceutically acceptable aerosols forinhalation. An example of a drug administered in the form of an aerosolis pentamidine which is administered to AIDS patients by inhalation toprevent pneumonia caused by Pneumocystis carnii.

The present invention further contemplates devices suitable fortransbronchial and transalveolar administration of PAIs. Such devicesinclude, but are not limited to, atomizers and vaporizers. The presentinvention also includes devices for electrical or direct injection.Electrical injection, or iontophoresis, is the process of using a smallelectrical current to drive charged elements, compounds and drugsthrough the skin for the purpose of delivering the therapeutic compoundto the local tissues or to the whole body without breaking the skin.

The above-mentioned compositions are meant to describe, but not limit,the methods of administering the PAIs of the invention. The methods ofproducing the various compositions are within the ability of one skilledin the art and are not described in detail here.

The methods of producing suitable devices for injection, topicalapplication, atomizers and vaporizers are known in the art and will notbe described in detail.

The invention further provides methods of treating apoptosis comprisingadministering an amount of the PAIs effective to inhibit apoptosis.Various apoptosis-related indications can be treated by the methodincluding, but not limited to, dermatological effects of aging,disorders and diseases, immunosuppression, gastrointestinalperturbations, cardiovascular disorders, rejection of tissuetransplantation, and Alzheimer's disease.

It has now been found that PAIs may be topically applied to the skin totreat a variety of dermatologic conditions. These conditions include,but are not limited to, wrinkling or sagging due to age and/orphotodamage, psoriasis. The present invention thus encompasses methodsof treating dermatological conditions. Furthermore, baldness may becaused by apoptosis of the cells of the hair follicles. Therefore, thePAIs would be suitable for use in topical treatment of the skin toprevent continued hair loss.

As discussed above, these conditions are preferably treated by topicalapplication of a composition comprising an effective amount of PAIs. Aneffective amount of PAI is one which ameliorates or diminishes thesymptoms of the dermatologic conditions. Preferably, the treatmentresults in resolution of the dermatologic condition or restoration ofnormal skin function; however, any amelioration or lessening of symptomsis encompassed by the invention.

Immunosuppression related disorders are caused by a variety of stimuliwhich include, but are not limited to, viruses including, but notlimited to, HIV, chemotherapeutic agents, and radiation. These stimulitrigger apoptosis in a variety of disorders, including, but not limitedto, those of the digestive tract tissues and associated gastrointestinalperturbations.

Gastrointestinal perturbations include, but are not limited to, damageto the lining of the gut, severe chronic ulcers, colitis, radiationinduced damage, chemotherapy induced damage, and the perturbation of thegastrointestinal tract caused by parasites, and diarrhea from any othercause. Various viral and bacterial infections are known to result ingastrointestinal perturbations; the PAIs are also suitable for use intreatment of the side effects associated with these infections. PAIs areparticularly suited for use in ameliorating the gastrointestinaldisturbances associated with chemotherapy. As shown in the Examplespresented below, mice treated with methotrexate and various PAIssuffered less feeding problems and had none of the diarrhea found in thecontrol animals. Thus, PAIs are suitable for use not only in preventingthe diarrhea associated with chemotherapy but also the nausea.

The PAIs are particularly suited to treatment of variousgastrointestinal conditions in animals, particularly cattle. Suchconditions, particularly diarrhea, account for the loss of many calves.Treatment of gastrointestinal conditions is preferably bygastrointestinal administration. In the case of cattle, an effectiveamount of the PAIs can be conveniently mixed in with the feed. Inhumans, administration can be by any method known in the art ofgastrointestinal administration.

In addition, the PAIs can be administered to immunodeficient patients,particularly HIV-positive patients, to prevent or at least mitigateapoptotic death of T cells associated with the condition, which resultsin the exacerbation of immunodeficiencies as seen in patients with fullblown AIDS. Preferably, administration of PAIs to such patients isparenterally, but may also be transdermal or gastrointestinally.

The PAIs can also be administered to treat apoptosis associated withreperfusion damage involved in a variety of conditions, including, butnot limited to, coronary artery obstruction; cerebral infarction;spinal/head trauma and concomitant severe paralysis; reperfusion damagedue to other insults such as frostbite; and any indication previouslythought to be treatable by superoxide dismutase (SOD).

Myocardial and cerebral infarctions are caused generally by a suddeninsufficiency of arterial or venous blood supply due to emboli, thrombi,or pressure that produces a macroscopic area of necrosis; the heart,brain, spleen, kidney, intestine, lung and testes are likely to beaffected. Apoptosis occurs to tissue surrounding the infarct uponreperfusion of blood to the area; thus, PAIs are effective ifadministered at the onset of the infarct, during reperfusion, or shortlythereafter.

Thus, the invention includes methods of treating apoptosis associatedwith reperfusion comprising administering a therapeutically effectiveamount of at least one PAI to a patient in need of such therapy.

The invention further encompasses a method of reducing the apoptosis andreperfusion damage associated with myocardial and cerebral infarctionsfor patients with a high risk of heart attack and stroke byadministering a therapeutically effective amount of at least one PAI toa patient in need of such therapy.

Preferably, treatment of reperfusion damage is by parenteraladministration of the compositions of the invention. Any other suitablemethod may be used, however, for instance, direct cardiac injection inthe case of myocardial infarct. Devices for such injection are known inthe art, for instance the Aboject cardiac syringe.

The invention further provides methods of limiting and preventingapoptosis in cells during the culture or maintenance of mammalianorgans, tissues, and cells by the addition of an effective amount ofPAIs to any media or solutions used in the art of culturing ormaintaining mammalian organs, tissues, and cells.

The invention further encompasses media and solutions known in the artof culturing and maintaining mammalian organs, tissues and cells, whichcomprise an amount of at least one PAI effective to limit or preventapoptosis of the cells in culture.

These aspects of the invention encompass mammalian cell culture mediacomprising an effective amount of at least one PAI and the use of suchmedia to limit or prevent apoptosis in mammalian cell culture. PAIs havebeen found to limit or prevent apoptosis under circumstances in whichcells are subjected to mild traumas which would normally stimulateapoptosis. Such traumas may include, but are not limited to, low levelirradiation, thawing of frozen cell stocks, rapid changes in thetemperature, pH, osmolarity, or ion concentration of culture media,prolonged exposure to non-optimal temperature, pH, osmolarity, or ionconcentration of the culture media, exposure to cytotoxins,disassociation of cells from an intact tissue in the preparation ofprimary cell cultures, serum deprivation (or growth in serum-freemedia).

Thus the invention encompasses compositions comprising tissue culturemedium and an effective amount of at least one PAI. Serum-free media towhich PAIs may be added as anti-apoptotic media supplements include, butare not limited to, AIM V® Media, Neuman and Tytell's Serumless Media,Trowell's T8 Media, Waymouth's MB 752/1 and 705/1 Media, andWilliams'Media E. In addition to serum-free media, suitable mammaliancell culture media to which PAIs may be added as anti-apoptotic mediasupplements include, but are not limited to, Basal Eagle's Media,Fischer's Media, McCoy's Media, Media 199, RPMI Media 1630 and 1640,Media based on F-10 & F-12 Nutrient Mixtures, Leibovitz's L-15 Media,Glasgow Minimum Essential Media, and Dulbecco's Modified Eagle Media.Mammalian cell culture media to which PAIs may be added further compriseany media supplement known in the art, including but not limited to,sugars, vitamins, hormones, metalloproteins, antibiotics, antimycotics,growth factors, lipoproteins and sera.

The invention further encompasses solutions for maintaining mammalianorgans prior to transplantation, which comprise an effective amount ofat least one PAI, and the use of such solutions to limit or preventapoptosis in such mammalian organs during their surgical removal andhandling prior to transplantation. In all cases concentrations of PAIsrequired to limit or prevent apoptosis can be determined empirically byone skilled in the art by methods like those found in Examples 2, 3 and4, as well as other methods known in the art.

It has also been found that the PAI fractions above a certainconcentration can form micelles in solution. The invention thus includescompositions comprising micelles.

The following examples are provided to illustrate but not limit theinvention.

EXAMPLE 1 PAI Isolation and Purification

Approximately 100 g of commercially available soybean flour (SigmaChemical Co. St. Louis, Mo. U.S.A. and Central Soya, Archer DanielMidlands) was suspended in 500 ml of 70% acetone and stirred at roomtemperature for 30 minutes. The delipidated soybean flour was recoveredby centrifugation at 1,500 g for 10 minutes. This material wasresuspended in 1 l of 50% ethanol and stirred at room temperature for 30minutes. The supernatant, the aqueous retentate, was reclaimed bycentrifugation at 1,500 g for 10 minutes.

The aqueous retentate was concentrated by ultrafiltration and theethanol was removed by diafiltration over a 10 kD membrane (AmiconBeverly Mass. U.S.A.). This material was then loaded directly ontoSepharose S100HR (Pharmacia Biotechnology, Inc. Piscataway, N.J.,U.S.A.) equilibrated in 10 mM ammonium bicarbonate. The peak of A₂₈₀absorbing material eluted in the void volume and was pooled andlyophilized.

The freeze-dried high molecular weight material was extracted into asingle phase mixture of chloroform: methanol:water (3:8:4) by adding thesingle phase mixture to the dried material and mixing at roomtemperature for 30 minutes. The mixture was then centrifuged to removethe insoluble material. The insoluble material yielded alipid/glycolipid fraction which retained PAI activity. This fraction hasbeen termed the L/G fraction. The carbohydrate composition of the L/Gfraction consists of arabinose and galactose in a 3:2 ratio with fucose,rhamnose, glucosamine, glucose and mannose all present as minorconstituents. The carbohydrate composition was determined as describedin Example 5.

The L/G fraction can be further separated on the basis of its solubilityin a mixture of chloroform:methanol (80:20) and chromatography on silica(Silicic Acid 100 mesh, Mallinckrodt Chemical, Inc. Kentucky). Thesilica chromatography is resolved in methanol to yield an activefraction (SiMe).

For a detailed summary of the physical and chemical characteristics ofthe soy flour extract at various stages of purification see Table 1,where ND stands for "none detected".

In Table 1, the activities and physical characteristics of the productsof four stages of purification were determined. These four stages were:aqueous retentate; 70% acetone extract; 50% ethanol extract of the 70%acetone pellet; and the high molecular weight fraction purified by sizeexclusion gel filtration chromatography from the 50% ethanol fraction.Protein yield is expressed as protein recovered per gram dry weight ofstarting material, as measured by the Bradford assay procedure (BioRadLaboratories). Anti-apoptotic activity is expressed as the calculatedconcentration of material (μg/ml of media) required to save 50% of thecells released on serum free treatment as described in Example 2.Trypsin inhibition is expressed in relative units per μg of protein inthe sample. A relative unit (U) was defined as the amount of inhibitoryactivity which decreases by 50% the initial rate of hydrolysis of a 100μM substrate by 2 μg of trypsin in a total volume of 1 ml. Absorbancevalues at 260 and 280 nm are expressed per gram of starting material ina 1 ml cell. This gives an indication of relative protein and nucleicacid concentrations present. The ratio of 260/280 was used to estimatethe amount of nucleic acid present as described in Dawson et al., Datafor Biochemical Research, Third Edition, 1986 published Oxford SciencePublications.

                                      TABLE 1                                     __________________________________________________________________________    Physical Characterization of Soy PAI Extract                                  at Various Stages of Purification                                                            Anti-                                                                 Protein apoptotic                                                                             Trypsin         Nucleic                                       Concentration                                                                         Activity                                                                              Inhibition                                                                          A280 A260 Acid                                   __________________________________________________________________________    Soy Flour                                                                            196 mg/gm                                                                             Inhibitory                                                                            0.561 U/μg                                                                       116/gm                                                                             134/gm                                                                             2.4%                                   Water                                                                         Extract                                                                       Soy Flour                                                                            140 μg/gm                                                                          Inhibitory                                                                            ND    4.5/gm                                                                             7.2/gm                                                                              11%                                   70% Acetone                                                                   Extract                                                                       Soy Flour                                                                            310 μg/gm                                                                          11  μg/ml                                                                            177 U/μg                                                                        32/gm                                                                              36/gm                                                                             ND                                     Acetone                                                                       Pellet                                                                        50% Ethanol                                                                   Extract                                                                       (AcE)                                                                         Soy Flour                                                                            62  μg/gm                                                                          0.14                                                                              μg/ml                                                                           3.5 U/μg                                                                         10/gm                                                                              12/gm                                                                             ND                                     Acetone                                                                       Pellet                                                                        50% Ethanol                                                                   Gel Filtra-                                                                   tion Pool                                                                     (FAcE)                                                                        Soy Flour                                                                            20  μg/gm                                                                          45  ng/ml                                                                             ND    8.4/gm                                                                             9.1/gm                                                                             ND                                     Acetone                                                                       Pellet                                                                        50% Ethanol                                                                   Gel Filtra-                                                                   tion Pool                                                                     Organic                                                                       Extract                                                                       (L/G)                                                                         Soy Flour                                                                            0.9 μg/gm                                                                          12  ng/ml                                                                             ND    6.55/gm                                                                            7.7/gm                                                                             ND                                     Acetone                                                                       Pellet                                                                        50% Ethanol                                                                   Gel Filtra-                                                                   tion Pool                                                                     Organic                                                                       Extract                                                                       Silicic acid                                                                  Methanol                                                                      Eluate                                                                        (SiMe)                                                                        __________________________________________________________________________

EXAMPLE 2 Apoptosis Assay with C3H 10T1/2 Cells

In order to determine the apoptotic activity of the PAIs, the followingexperiment was performed. The cell assay is described in PCT/US94/04942.Briefly, the cells, C3H 10T1/2 clone 8, were assayed at confluence (FIG.1), during exponential growth phase when cell cycle position is randomlydistributed with no cells arrested in G_(o) (FIGS. 2 and 3), and inquiescence (FIG. 4). Exponential growth phase was assured by seeding at2000 cells per 1 ml (5 ml for a 60 mm culture plate) five days prior tothe beginning of the experiment. At T=0, cultures were transferred toserum-free medium, as an apoptosis stimulus, and seed extracts wereadded. Controls included 10⁻⁷ M 12-O-tetradecanoyl phorbol-13-acetate(TPA) to ensure the responsiveness of the cell culture. The PAI samplesprepared by extraction with ethanol and by gel filtration were added toserum free medium at 0.1 g dry weight equivalents and sterile filteredprior to addition to the cultures. Assays were performed in triplicateor quadruplicate. Analyses of the cell responses were made after between22 and 28 hours of serum deprivation and/or treatment with soy flourderived PAIs. Two assays were performed on each cell culture plateconsisting of differential cell counts.

1. All non-adherent or loosely adherent cells were removed from theculture dish and counted by appropriate techniques, typically countingby electric particle counting instrument. These are the apoptotic cells,the serum deprived released cells (SDR), released by the action ofcultivation in serum-free medium. Approximately 95% of these releasedcells are apoptotic as shown by both ultrastructure analysis and DNAfragmentation analysis.

2. The remaining adherent cells (ADH) are exposed to a buffered,typically pH 7.3, balanced salt solution such as Hanks Balanced SaltSolution without calcium and magnesium salts containing 0.05% trypsinand 0.53 mM ethylenediaminetetraacetic acid (EDTA). Each culture isincubated at either room temperature or 37° C. on a rocking platform toensure uniform distribution of the trypsin reagent over the culturesurface. After a standardized period of time, typically 10 minutes, thereleased cells are removed from each culture dish and measured by thesame means as described above, typically electronic particle counting.This ADH cell count is comprised of both trypsin resistant and trypsinsensitive cells as described PCT/US94/04942.

The results obtained from the apoptosis cell assays are presented inFIGS. 1, 2, 3 and 4. In FIG. 1 the percentage of cells having undergoneapoptosis (SDR) and adherent cells (ADH) are presented separately. TheData in FIG. 1 demonstrate that the PAIs are effective in reducingapoptosis in confluent cells, as compared with the Basal Medium Eagle(BME), serum-deprived control.

In FIG. 2, the results are presented as a percentage of adherent cellsin the samples treated with PAIs normalized for the number of adherentcells in the serum free control sample without PAIs. In other words, thepercentage of cells saved from apoptosis by treatment with PAIs. Thedata presented in FIG. 2 demonstrate that soy PAIs have aconcentration-dependent anti-apoptotic effect on C3H 10T1/2 cells inexponential growth phase.

In FIG. 3, the anti-apoptotic activity for PAIs after extraction with50% ethanol (right side) and PAIs which have been further purified bysize exclusion gel filtration chromatography (left side) is presented.The results in FIG. 3 are expressed as the percentage of cells savedfrom apoptosis (SDR cells converted to ADH cells) by treatment with PAIsas compared with the no PAI serum free control samples, all of which isexpressed as a function of trypsin inhibitory units. The data presentedin FIG. 3 demonstrate that when the concentration of Bowman-Birkinhibitors (as measured by trypsin inhibition) is reduced by sizeexclusion gel filtration chromatography, the anti-apoptotic activity ofPAIs is maintained. Thus, the anti-apoptotic activity of the PAIpreparations is not due to the presence of Bowman-Birk inhibitors.

In FIG. 4, the anti-apoptotic activity of various concentrations of soyPAIs on quiescent C3H 10T1/2 cells treated with cycloheximide ispresented. Quiescent cells are those which no longer respond to serumdeprivation by entering apoptosis. Rather, apoptosis is stimulated inthese cells by the addition of 10 μg/ml cycloheximide in C3H 10T1/2.Typically, these cells become confluent after about one week in cultureand quiescent after about two weeks in culture. The results in FIG. 4are expressed as viable cells remaining (ADH) after a given treatment.The data in FIG. 4 demonstrate that soy PAIs have a small, butsignificant anti-apoptotic effect on quiescent C3H 10T1/2 cells.

EXAMPLE 3 Apoptosis Assay with Neonatal Rat Cardiac Myocytes

Myocytes were prepared from hearts of day-old rats as described inSimpson Circulation Research (1985) 56:884-894. In brief, the individualcells were obtained by brief, alternating cycles of room temperaturetrypsinization and mechanical disaggregation. The cells were collected,washed, and resuspended in MEM, 5% fetal bovine serum and 50 U/mlpenicillin-G. To reduce contamination by non-myocytes, the cells werepre-plated for 30 minutes. The non-adherent cardiac myocytes wereremoved from the culture dish, counted on a hemocytometer, andresuspended in medium to a concentration of 600,000 viable cells/ml. Thecell suspension was distributed into different culture dishes andincubated in a 37° C., 5% CO₂ incubator for 16-24 hours. The yield was3-5×10⁶ cells/heart and viable cells were >90% by trypan blue staining.

On the first day of culture, the cells were rinsed with Minimal EagleMedium (MEM) several times to remove debris and non-adherent cells. Theywere replenished with serum supplemented media as above. The myocyteswere challenged with different conditions in RPMI 1640 medium the nextday. The results obtained are presented in Table 2, where PAI 1×represents the material obtained from 0.1 g of soy flour startingmaterial.

                  TABLE 2                                                         ______________________________________                                                      Neonatal Cardiomyocytes                                                       Beating Rate                                                                           Cell Number                                            ______________________________________                                        Serum-free      +          11606                                              Conditioned Media                                                                             +++++       5128                                              PAI 1×    +++        15062                                              ______________________________________                                    

The results obtained indicate that the PAI fraction is capable ofpreserving the well-being of cells in the presence of anapoptosis-inducing insult.

EXAMPLE 4 Determination of Carbohydrate Composition of PAI

In order to determine whether PAIs contain carbohydrate, the L/Gfraction of PAIs were exposed to various conditions and the resultingcarbohydrate residues were assayed. The PAIs were obtained from SigmaSoy flour Lot No. 103H0820 treated to obtain PAIs as described inExample 1. The free monosaccharides in untreated PAIs were determined byHPLC on Dionex Carbopac™ PA1 in 16 mM NaOH according to the methoddescribed in Dionex Document No. 034441. The results obtained arepresented in Table 3. The sample was then hydrolyzed with 2N TFA forfour hours at 100° C. as described by Hardy and Townsend, (1994) Meth.Enzymol. 230:208-225. The results obtained are presented in Table 4. Thesample was further hydrolyzed with 6N HCl for 6 hours at 100° C. asdetermined by the method described by Hardy and Townsend (1994). Theresults obtained are depicted in FIG. 5.

                  TABLE 3                                                         ______________________________________                                        Monosaccharides Detected in PAI                                               Monosaccharide                                                                              ng/25 μg PAI                                                                           ng/mg PAI                                           ______________________________________                                        Arabinose     1.098       45.9                                                Glucose       0.058        2.36                                               TOTAL         1.157        48.26                                              ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Monosaccharides Released on Hydrolysis                                        Monosaccharide                                                                              ng/50 μg PAI                                                                           μg/mg PAI                                        ______________________________________                                        Fucose        1625        32.50                                               Arabinose     4605        92.10                                               Galactosamine  86          1.71                                               Glucosamine    478         9.56                                               Galactose     5280        105.60                                              Glucose        352         7.04                                               Xylose         520        10.40                                               TOTAL         12946       258.91                                              ______________________________________                                    

EXAMPLE 5 Chemical Cleavage of PAIs and Anti-Apoptotic Activity ofResulting PAI Constituents

Three separate samples of PAI obtained by the method described inExample 1 were treated as described in the protocols of three differentkits produced by Oxford GlycoSystems, Inc., Cross Island Plaza, 133-33Brookville Boulevard, Rosedale, N.Y. 11422.

One PAI sample was treated with the reagents of Oxford GlycosystemsGlycofree™ Deglycosylation Kit as described by the manufacturer'sinstructions. This kit is based on a modification of the use ofanhydrous trifluoromethane sulfonic acid (TFMS) to deglycosylateglycoproteins as described by Sojar and Bahl (1987) Meth. Enzymol.138:341-350. Specifically the sample was treated as follows. An aliquotof PAI was 1) lyophilized; 2) incubated with TFMS; 3) excess TFMS wasneutralized; and 4) the sample was treated to recover deglycosylatedPAIs. All steps were performed as described by or in accordance with themanufacturer's instructions.

A second PAI sample was treated with the reagents of Oxford GlycosystemsN-Glycan Recovery Kit as described by the manufacturer's instructions.This kit is based on the use of anhydrous hydrazine to cleaveN-Glycosidic bonds as reported by Takasaki et al. (1982). Specifically,the sample was treated as follows: 1) lyophilized; 2) treated withanhydrous hydrazine at 95° C. for 5 hours to cleave N-glycosidic bonds;3) subjected to chromatography to remove unreacted hydrazine and peptidematerial; 4) treated with acetic anhydride to re-N-acetylate anyde-N-acetylated amino groups; 5) a trace of Cu²⁺ ion was added to thesample to convert the acetohydrazone derivatives to unreducedoligosaccharides; 6) subjected to mixed-bed ion-exchange chromatographyto remove Cu²⁺ ion and monoacetyl hydrazine; and 7) the unreducedN-glycans of PAI, substantially free of peptide material, were recoveredin a dilute solution of acetic acid. All the steps were performed asdescribed by or in accordance with the manufacturer's instructions.

A third PAI sample was treated with the reagents of the OxfordGlycosystems O-Glycan Recovery Kit as described by the manufacturer'sinstructions. Specifically the sample was treated as follows: 1)lyophilized; 2) treated with anhydrous hydrazine at 60° C. for 5 hoursto cleave O-glycosidic bonds; 3) subjected to chromatography to removeunreacted hydrazine and peptide material; 4) treated with aceticanhydride to re-N-acetylate any de-N-acetylated amino groups; 5) a traceof Cu²⁺ ion was added to the sample to convert the acetohydrazonederivatives of the sample to unreduced oligosaccharides; 6) subjected tomixed-bed ion-exchange chromatography to remove Cu²⁺ ion and monoacetylhydrazine; and 7) the unreduced O-glycans of PAI, substantially free ofpeptide material, were recovered in a dilute solution of acetic acid.All steps were performed as described by or in accordance with themanufacturer's instructions.

The anti-apoptotic activity of each of the three aforementioned sampleswas tested and the results are reported in Table 5.

                  TABLE 5                                                         ______________________________________                                        PAI Constituent   Anti-Apoptotic Activity                                     ______________________________________                                        Deglycosylated    None                                                        PAI Protein                                                                   PAI-Derived       Yes                                                         N-Glycans                                                                     PAI-Derived       None                                                        O-Glycans                                                                     ______________________________________                                    

EXAMPLE 6 Use of PAIs to prevent chemotherapy induced gastrointestinaldisorders

In order to determine the in vivo activity of the PAIs, the followinganimal experiments were performed. In Examples 6 and 7 the animal testswere performed essentially as described in Funk and Baker (1991) J.Nutr. 121:1684-1692; and Funk and Baker (1991) J. Nutr. 121:1673-1683.Briefly, male Sprague-Dawley rats were used to determine if isolated AcEand L/G obtained from soy flour as described in Example 1 couldalleviate methotrexate (MTX) toxicity. The rats were housed inindividual, wire-bottom stainless steel cages and were adapted to theirrespective diets for 7 days prior to injection of MTX and remained onthe same diets for 7 days after injection. Diets fed were semipurifiedrat food with the following additions:

1. casein

2. casein and soy concentrate (10 g and 10 g)

3. casein and soy flour (10 g and 10 g)

4. casein and AcE

5. casein and L/G

AcE and L/G were used at concentrations equal to that extracted from thesoy starting material.

Records of rat weight and food intake were kept during the preinjectionperiod. Rats were injected IP with 20 mg/kg MTX. During the 7 daypostinjection period, rat weight, food intake and incidence of diarrheawere recorded. Rat weight and food intake data were analyzed using thenonparametric Kruskal-Wallis test and post-hoc comparison. The P valuewas adjusted for multiple comparisons by dividing 0.05 by the number ofcomparisons made (10). A nonparametric test was used because variancesbetween groups were not homogeneous and thus assumptions for analysis ofvariances were not met. Food intake following MTX injection wasexpressed as a percentage of the average intake 3 days prior toinjection for each animal. Thus, each animal served as its own control.Only days 3, 4, 5, and 6 post MTX injection were analyzed statistically.The reason for this is that days 3 and 4 are when toxicity is mostsevere and days 5 and 6 are when recovery begins. Diarrhea data wereanalyzed using both the Fisher's Exact Test and loglinear analysis.

Results showed that soy concentrate and soy flour starting material aswell as both the AcE and L/G were capable of improving food intakefollowing MTX injection (Table 6 and FIG. 7). On day 3 food intake forrats consuming soy flour and soy concentrate was statistically greaterthan for those consuming casein alone or casein with L/G. Rats consumingcasein with AcE were intermediate in food intake on day 3 andstatistically similar to all groups except those consuming soy flour.Day 4 showed an identical pattern except since food intake for ratsconsuming L/G rose slightly as compared with day 3, these rats were nolonger statistically different from rats consuming soy concentratealthough numerically food intake remained substantially lower. Recoverywas evident on days 5 and 6 and food intake was statistically similaramong all groups. Weight change (Table 7) reflected patterns observed infood intake which is expected. Rats consuming soy concentrate and soyflour gained a substantial amount of weight during the first 4 daysfollowing MTX injection. Rats consuming AcE, L/G or casein alone gainedless weight and those consuming casein gained statistically less thanthose fed soy concentrate or soy flour. Differences in incidence ofdiarrhea were not statistically different but the pattern of diarrheawas consistent with food intake and weight change (Table 7). Ratsconsuming soy concentrate or soy flour had no diarrhea while a slightamount of diarrhea was present in rats consuming AcE (10%) and amoderate amount of diarrhea was present in those consuming L/G or caseinalone (30-40%).

In conclusion, this experiment showed that soy concentrate and soy flouroffered the best protection of the components tested. Casein with AcEappeared to be intermediate and superior to casein alone or L/G asevidenced by better maintenance of food intake and weight and lowerincidence of diarrhea. This result indicates that compounds isolatedfrom soy can provide protection against MTX toxicity. In thisexperiment, the L/G fraction at this concentration did not appear toprovide protection. However, Example 8 shows that increasedconcentrations of L/G are effective.

                                      TABLE 6                                     __________________________________________________________________________    Effect of Diet and Methotrexate on Food Intake.sup.1                                          Pretreatment                                                                  Food Intake.sup.2                                                                    Food Intake Posttreatment (%).sup.3                    Diet            (g/day)                                                                              day 3  day 4  day 5  day 6                             __________________________________________________________________________    Casein          18.4 ± 0.6                                                                        34.1 ± 11.0.sup.a                                                                 39.6 ± 13.0.sup.a                                                                 83.0 ± 12.5                                                                       108.9 ± 2.7                    Soy Concentrate-Casein (50/50)                                                                18.4 ± 0.5                                                                        94.3 ± 6.5.sup.bc                                                                 94.8 ± 5.1.sup.bc                                                                 98.9 ± 2.5                                                                        101.8 ± 3.3                    Soy Flour-Casein (50/50)                                                                      17.8 ± 0.4                                                                        99.0 ± 4.6.sup.c                                                                  99.1 ± 2.0.sup.c                                                                  98.2 ± 2.3                                                                        102.1 ± 3.5                    Casein-AcE      18.5 ± 0.4                                                                        63.6 ± 9.3.sup.ab                                                                 68.3 ± 10.3.sup.ab                                                                88.6 ± 6.9                                                                         99.5 ± 4.0                    Casein-L/G      19.0 ± 0.8                                                                        46.3 ± 11.7.sup.a                                                                 53.9 ± 14.2.sup.ab                                                                75.3 ± 13.1                                                                        96.4 ± 11.0                   __________________________________________________________________________     .sup.1 Values are means ± standard error of the mean for ten male rats     Methotrexate was injected IP following a 7day adaptation period. Values i     columns with unlike superscripts differ (P ≦ 0.05, KruskalWallis       test and post hoc comparison).                                                .sup.2 Pretreatment food intake represents the mean of the 3 day period       prior to the administration of MTX.                                           .sup.3 Posttreatment intake represents the % of pretreatment intake.     

                                      TABLE 7                                     __________________________________________________________________________    Effect of Diet and Methotrexate on Rat Weight.sup.1                                           Average Pretreatment                                                                     Weight Change                                                                             Incidence of                                           Weight.sup.2                                                                             day 0-4                                                                             day 4-6                                                                             Diarrhea                               Diet            (g)        (g)   (g)   (g)                                    __________________________________________________________________________    Casein          230.0 ± 4.7                                                                           .sup. 1.2 ± 5.3.sup.a                                                            16.3 ± 1.5                                                                       30                                     Soy Concentrate-Casein (50/50)                                                                227.2 ± 4.7                                                                           22.1 ± 1.5.sup.b                                                                  9.7 ± 1.6                                                                       0                                      Soy Flour-Casein (50/50)                                                                      227.2 ± 4.4                                                                           22.7 ± 1.6.sup.b                                                                 10.1 ± 1.0                                                                       0                                      Casein-AcE      230.3 ± 3.2                                                                            11.7 ± 4.1.sup.ab                                                               13.5 ± 2.1                                                                       10                                     Casein-L/G      232.9 ± 5.1                                                                             6.9 ± 5.6.sup.ab                                                                9.8 ± 4.5                                                                       40                                     __________________________________________________________________________     .sup.1 Values are means ± standard error of the mean for ten male rats     Methotrexate was injected IP following a 7day adaptation period. Values i     columns with unlike superscripts differ (P ≦ 0.05, KruskalWallis       test and post hoc comparison).                                                .sup.2 Pretreatment weight indicates the average weight on the day of         injection.                                                               

EXAMPLE 7 Use of PAIs to prevent chemotherapy induced gastrointestinaldisorders

Male Sprague-Dawley rats were used to determine if graded levels ofisolated soy fractions (ACE, L/G and MAcE) could alleviate methotrexate(MTX) toxicity. Animals were housed in individual, wire-bottom stainlesssteel cages. Rats were adapted to their respective diets for 7 daysprior to injection of MTX and remained on the same diets for 7 daysafter injection. Diets fed were semipurified and casein with additionsas follows:

1. No additions

2. AcE (100 mg/20 g casein; 1×)

3. AcE (300 mg/20 g casein; 3×)

4. AcE (1000 mg/20 g casein; 10×)

5. L/G (10 mg/20 g casein; 1×)

6. L/G (30 mg/20 g casein; 3×)

7. L/G (100 mg/20 g casein; 10×)

8. MAcE (100 mg/20 g casein; 1×)

9. MAcE (300 mg/20 g casein; 3×)

10. MAcE (1000 mg/20 g casein; 10×)

11. MAcE (3000 mg/20 g casein; 30×)

Note that MAcE is soy molasses extracted as for soy flour to obtain AcE.Each diet group contained 8 rats except for the group receiving caseinwith no added compound which contained 10 rats. Records of rat weightand food intake were kept during the preinjection period. Rats wereinjected IP with 20 mg/kg MTX. During the 7 day postinjection period,rat weight, food intake and incidence of diarrhea were recorded. Foodintake for various groups is depicted in FIGS. 8-10. Incidence ofdiarrhea is depicted in FIG. 11. Rat food intake for the entire group isdepicted in FIG. 12. Rat weight and food intake data were analyzed byANOVA using a factorial arrangement of treatments to test the maineffects of compound and dose and the possible interaction betweencompound and dose. Factorial analysis was done using only the treatmentgroups with the 1×, 3× and 10× doses of each of the compounds. Inaddition t-tests were used to determine differences between the 10×level of each compound and the diet containing only casein. Food intakefollowing MTX injection was expressed as a percentage of the averageintake 3 days prior to injection for each animal. Thus, each animalserved as its own control. Only days 3, 4, 5 and 6 post MTX injectionwere analyzed statistically. The reason for this is that days 3 and 4are when toxicity is most severe and days 5 and 6 are when recoverybegins. Diarrhea data were analyzed using Fisher's Exact Test. Only the10× levels for each of the compounds were analyzed statistically againstcasein for differences in diarrhea incidence. FIG. 12. The reason forthis is that Fisher's test is a conservative test. When multiplecomparisons are done the error rate must be adjusted. So in order toincrease the chances of statistical significance, only those comparisonswere done where the best response for each of the compounds had beenrealized as evidenced by food intake and weight change data.

Results of the factorial analysis of food intake and weight change arepresented in Tables 8 and 9. The results showed that the AcE compoundwas the most effective at alleviating MTX toxicity. Food intake wasgreater for all AcE groups combined than for both of the other groups onday 3 following MTX dosing and remained greater than those consumingMAcE on day 4 (P<0.05). Decreased toxicity in rats consuming AcE ascompared with MAcE was also reflected in weight patterns as thoseconsuming AcE gained more weight during the first four days postdosingthan those consuming MAcE (P<0.05). Improvements in intake andmaintenance of weight were seen with increasing levels of each of thecompounds with the exception of the 30× level of MACE, although this wasnot statistically significant. The level of each compound where responsewas the best was 10×. In comparing the 10× level of each of thecompounds against casein alone, AcE was statistically greater on days 3,4 and 5 postdosing (P<0.05). The pattern of diarrhea was consistent withthe food intake results. Fifty percent of the animals consuming caseindeveloped diarrhea. No animals consuming the 10× level of the compoundsdeveloped diarrhea which was statistically less than those consumingcasein alone (P=0.088). All other groups experienced some diarrhea withthe exception of those consuming the 3× level of AcE.

In conclusion, for the compounds tested, AcE was the best at alleviatingMTX toxicity. L/G and MAcE positively affected MTX toxicity as evidencedby decreased incidence of diarrhea as compared with casein alone andstatistically nonsignificant improvement in food intake and weightmaintenance. It is possible that higher levels of AcE and L/G mayprovide additional protection. The 30× level of MAcE proved to beineffective and closely resembled casein alone. Therefore, it appearsthat once a threshold is reached higher levels are detrimental. It ispossible, that MAcE may be more effective at a dose somewhere betweenthe 10× and 30× levels tested in this experiment.

                                      TABLE 8                                     __________________________________________________________________________    Effect of Diet and Methotrexate on Food Intake.sup.1                               Pretreatment                                                                  Food Intake                                                                          Food Intake Posttreatment (%)                                     Diet                                                                             n (g/day)                                                                              day 3.sup.2,3                                                                        day 4.sup.3,4                                                                       day 5.sup.3                                                                         day 6                                          __________________________________________________________________________    1. 10                                                                              18.5 ± 0.5                                                                         39.5 ± 11.5                                                                      33.5 ± 11.7                                                                      58.9 ± 12.4                                                                       96.3 ± 5.9                                 2. 8 19.7 ± 0.6                                                                         57.6 ± 12.8                                                                      52.7 ± 16.6                                                                      71.5 ± 11.2                                                                       93.0 ± 4.8                                 3. 8 16.2 ± 0.4                                                                        61.4 ± 7.0                                                                        71.3 ± 11.3                                                                      92.6 ± 6.0                                                                       105.6 ± 4.7                                 4. 8 16.7 ± 0.4                                                                        78.2 ± 9.2                                                                        76.7 ± 11.0                                                                      90.3 ± 6.7                                                                        97.8 ± 4.3                                 5. 8 17.9 ± 0.4                                                                        40.0 ± 8.4                                                                        45.9 ± 11.4                                                                      80.3 ± 11.4                                                                      105.7 ± 3.6                                 6. 8 18.3 ± 0.7                                                                        46.0 ± 9.8                                                                        50.7 ± 12.8                                                                      79.6 ± 12.0                                                                      105.2 ± 5.3                                 7. 8 17.3 ± 0.6                                                                        60.8 ± 9.9                                                                        61.9 ± 12.8                                                                      79.2 ± 11.8                                                                      103.3 ± 3.9                                 8. 8 18.5 ± 0.7                                                                         40.8 ± 10.2                                                                      34.5 ± 9.4                                                                       59.1 ± 9.8                                                                       100.2 ± 4.8                                 9. 8 17.2 ± 0.5                                                                        38.0 ± 8.4                                                                        45.4 ± 10.6                                                                      82.2 ± 11.7                                                                      100.4 ± 6.4                                 10.                                                                              8 16.9 ± 0.4                                                                        50.4 ± 5.8                                                                        52.2 ± 9.4                                                                       70.4 ± 13.9                                                                       89.3 ± 12.0                                   8 16.4 ± 0.4                                                                        30.0 ± 6.0                                                                        33.9 ± 9.8                                                                       58.1 ± 9.1                                                                       110.4 ± 4.0                                 __________________________________________________________________________     Footnotes:                                                                    .sup.1 Values are means ± standard error of the mean for male rats.        Methotrexate was injected IP following a 7day adaptation period.              Pretreatment food intake represents the mean of the 3day period prior to      the administration of methotrexate. Posttreatment intake represents the %     of pretreatment intake.                                                       .sup.2 The caseinAcE groups (2-4) maintained a better posttreatment intak     than caseinL/G (56) and caseinMAcE (8/11) groups (P < 0.05, ANOVA and         Student NewmansKeuls tests following factorial analysis).                     .sup.3 CaseinAcE (10X) (3) animals had a greater posttreatment intake tha     casein (P < 0.05), ttest).                                                    .sup.4 The caseinAcE groups maintained a better posttreatment intake than     the caseinMAcE groups (P < 0.05, ANOVA and Student NewmansKeuls tests         following factorial analysis).                                           

                  TABLE 9                                                         ______________________________________                                        Effect of Diet and Methotrexate on Rat Weight and Diarrhea.sup.1                         Average                    Incidence                                          Pretreatment                                                                             Weight Change   of Diar-                                           Weight     day 0-4.sup.2                                                                          day 4-6  rhea.sup.3                            Diet n     (g)        (g)      (g)      (%)                                   ______________________________________                                        1.   10    220.2 ± 5.5                                                                           -2.1 ± 1.8                                                                          13.2 ± 1.8                                                                          50                                    2.   8     233.5 ± 4.2                                                                           8.3 ± 5.6                                                                           11.1 ± 0.7                                                                          25                                    3.   8     224.7 ± 3.6                                                                           9.0 ± 3.0                                                                           10.8 ± 2.0                                                                          0                                     4.   8     227.3 ± 3.8                                                                           12.4 ± 5.4                                                                          10.6 ± 1.5                                                                          0                                     5.   8     232.6 ± 2.7                                                                           2.5 ± 3.6                                                                           14.3 ± 1.9                                                                          12.5                                  6.   8     225.5 ± 3.3                                                                           3.8 ± 3.9                                                                           12.2 ± 1.8                                                                          12.5                                  7.   8     232.6 ± 4.5                                                                           6.3 ± 5.0                                                                           11.3 ± 1.2                                                                          0                                     8.   8     234.1 ± 6.3                                                                           0.0 ± 2.3                                                                           13.5 ± 2.4                                                                          12.5                                  9.   8     226.1 ± 3.2                                                                           1.5 ± 4.9                                                                           11.6 ± 1.7                                                                          25                                    10.  8     235.2 ± 2.3                                                                           3.1 ± 3.2                                                                            7.9 ± 3.8                                                                          0                                     11.  8     220.6 ± 2.8                                                                           -2.3 ± 3.4                                                                          140.0 ± 1.2                                                                         25                                    ______________________________________                                         Footnotes:                                                                    .sup.1 Values are means ± standard error of the mean for male rats.        Methotrexate was injected IP following a 7day adaptation period.              Pretreatment weight indicates the average weight on the day of injection.     .sup.2 The caseinAcE groups (2-4) maintained overall weight better during     acute toxicity than the caseinMAcE groups (8-11) (P < 0.05, ANOVA and         Student NewmansKeuls tests following factorial analysis). CaseinAcE (10X)     (4) animals showed significantly less weight loss than casein (P < 0.05),     ttest).                                                                       .sup.3 Animals consuming the 10X level of the compounds (4, 7, 10) had a      significantly lower incidence of diarrhea than animals consuming casein (     < 0.088, Fisher's exact test for diarrhea).                              

EXAMPLE 8 Use of PAIs to inhibit apoptosis in lymphocytes obtained froman HIV-infected patient

The L/G fraction of PAIs isolated from soy flour was tested for itsability to inhibit apoptosis in lymphocytes from an HIV-infectedpatient.

Peripheral blood monocytes (PBMCs) were obtained from the patient andisolated according to standard methods. The PBMCs were cultured at 2×10⁶/well in 24-well plates (Costar--Cambridge, Mass.) for 72 hours at 37°C. and 5% CO₂ containing 2 ml/well of RPMI 1640 with antibiotics and 10%hAB. Some cultures contained 10 μg/ml Pokeweed Mitogen (PWM) (Sigma, St.Louis, Mo.). Suspensions of thymocytes were used immediately afterremoval or after culture in RPMI+10% fetal bovine serum with 5 μMdexamethasone (DEX) (Sigma--St. Louis, Mo.) for 18 hours. The cells wereexposed for three days to the L/G fractions at the concentrationsindicated below where 0.5 gEQ is the fraction derived from 0.5 gstarting weight of flour.

    ______________________________________                                        Lane #  PWM         L/G                                                       ______________________________________                                        1       -           None                                                      2       -           Purified L/G - 0.5 gEQ/ml                                 3       -           Purified L/G - 0.05 gEQ/ml                                4       -           Purified L/G - 0.005 gEQ/ml                               5       +           None                                                      6       +           Purified L/G - 0.5 gEQ/ml                                 7       +           Purified L/G - 0.05 gEQ/ml                                8       +           Purified L/G - 0.005 gEQ/ml                               9       -           Untreated mouse thymocytes (negative                                          control)                                                  10      -           Dexamethasone-treated mouse thymocytes                                        (positive control)                                        11      -           123 bp DNA standards                                      ______________________________________                                    

DNA was extracted and gel electrophoresis was performed as described bySambrook et al. Molecular Cloning--Laboratory Manuals, 2nd Ed. ColdSpring Harbor Laboratory Press, N.Y. pp. 134-135, E3-E4 and E10-11.Briefly, cells harvested were pelleted by centrifugation and lysed in400 μl of 50 mM KCl, 10 mM Tris-HCl (pH 8), 1% NP-40, 1% Tween-20, and0.5 mg/ml Proteinase K (Boehringer Mannheim, Indianapolis, Ind.) at 60°C. for 1 hour. After extraction with phenol-chloroform and recovery withethanol, the DNA was run through a 1.5% agarose gel (SeaKem, Rockland,Me.) in 90 mM Tris-Borate, 2.5 mM EDTA, pH 8.3 at 30 to 50 V forapproximately 4 hours. A 123 base pair ladder (GIBCO BRL, Gaithersburg,Md.) was used as the DNA standard (DS) markers. Gels were stained with 1g/ml ethidium bromide (Molecular Probes, Eugene, Ore.) and destained indistilled H₂ O.

The results are shown in FIG. 13 where the lanes are as indicated above.

The results obtained indicate that significant DNA fragmentation wasobserved in the absence of PWM-stimulation (lane 1) and thisfragmentation was almost completely inhibited in cultures that contained0.5 gEQ of L/G (lane 2). Lower concentrations of L/G (0.05 gEQ and 0.005gEQ) did not inhibit DNA fragmentation in the absence of PWM (lanes 3and 4) or in the presence of PWM (lanes 7 and 8). The DNA fragmentationin the presence of PWM (lane 5) was increased in comparison to cultureswithout PWM (lane 1). A slight inhibition of DNA fragmentation in thepresence of PWM was observed in the presence of 0.5 gEQ L/G (lane 6) incomparison to lane 5. Negative and positive controls (lanes 10 and 11)worked as expected.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced. Therefore, thedescriptions and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

We claim:
 1. A method of preventing apoptosis in cells during theculture or maintenance of mammalian organs, tissues and cells comprisingtreating the cells with a composition comprising a tissue culture mediaand an effective amount of at least one phytogenic apoptosis inhibitor.2. The method according to claim 1, wherein the cells are mammalian. 3.The method according to claim 2, wherein the cells are human.
 4. Themethod according to claim 1, wherein the cells are part of a tissue. 5.The method according to claim 1, wherein the cells are part of an organ.